Materials and Reagents

MicroAmp^TMSplash-Free 96-Well Base (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4312063 )
1,000 µl pipette tips (Corning, Axygen^®, catalog number: TF-1000-R-S )
200 µl pipette tips (Corning, Axygen^®, catalog number: TF-200-R-S )
10 µl pipette tips (Corning, Axygen^®, catalog number: TF-300-R-S )
1 ml needless syringe (BD, catalog number: 309659 )
1.5 ml RNase-free tube (Corning, Axygen^®, catalog number: MCT-150-C )
Axygen^® 0.2 ml Polypropylene PCR Tube Strips (8-Tubes/Strip) (Corning, Axygen^®, catalog number: PCR-0208-C )
MicroAmp^TM Optical 96-Well Reaction Plate with Barcode (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4306737 )
MicroAmp^TM Optical Adhesive Film (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4360954 )
Tissue paper
Healthy 5-6 weeks N. benthamiana plants (see Figure 1)
Any PAMPs of interest and corresponding control solution
Liquid nitrogen
β-mercaptoethanol (Solarbio, catalog number: M8210 )
E.Z.N.A.^® Total RNA Kit I (Omega Bio-Tek, catalog number: R6834-01 )
DNase/RNase-free ddH₂O (Solarbio, catalog number: R1600 )
PrimeScript^TMRT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio, Clontech, catalog number: RR047A )
TB Green^TM Premix Ex Taq^TM (Tli RNase H Plus) (Takara Bio, Clontech, catalog number: RR420A )
1 mM flg22 stock solution (Gene Script, RP19986) (see Recipes)
1 mM XEG1 stock solution (see Recipes)
70% ethanol (see Recipes)

Equipment

Mortar and pestle
Growth chamber
NanoDrop^TM1000 spectrophotometer (Thermo Fisher Scientific, model: NanoDrop^TM 1000 )
Vortexer (Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0246)
Cold Centrifuge (Eppendorf, model: 5424 R , catalog number: 5404000014)
Pipettes 100-1,000 µl (Eppendorf, catalog number: 3120000062 )
Pipettes 10-100 µl (Eppendorf, catalog number: 3120000046 )
PCR Thermal Cyclers (Thermo Fisher Scientific, Applied Biosystems^TM, model: 2720, catalog number: ED000651 )
MicroAmp^TM Adhesive Film Applicator (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4333183 )
MPS 1000 Mini PCR Plate Spinner (Labnet International, catalog number: C1000 )
ABI 7500 fast real-time PCR system (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4351107 )

Software

Applied Biosystems Sequence Detection Software v1.4.0

Procedure

Infiltration of Nicotiana benthamiana
1. N. benthamiana plants were grown in a growth chamber at 19-22 °C under long-day conditions (14-h-light/10-h-dark) and 70-80% relative humidity for 5-6 weeks as shown in Figure 1.
  
  Figure 1. Nicotiana benthamiana plant used for PAMP treatment
2. N. benthamiana leaves of the same position and similar size were treated with PAMPs (e.g., flg22) or control (e.g., ddH₂O) by infiltration using 1 ml needleless syringe (Figure 2). The infiltrated plant were dried gently using the tissue paper and kept in the growth chamber for 3-6 h before collected and frozen in liquid nitrogen.
  
  Figure 2. Infiltration of N. benthamiana using the needless syringe
Total RNA isolation
Total RNA was isolated from N. benthamiana leaves using E.Z.N.A.^® Total RNA Kit I isolation kit according to the manufacturer’s instructions with proper modifications.
1. Harvest the N. benthamiana leaves at 3-6 h after PAMP treatment. Grind the frozen tissues into powder with a mortar and pestle in liquid nitrogen.
2. Transfer the leaf powder into the 1.5 ml RNase-free tube with 700 μl TRK Lysis Buffer and 14 μl β-mercaptoethanol, vortex the samples for 30 sec, and maintain the sample at 4 °C for 5-10 min.
3. Centrifuge the samples at 15,000 x g for 5-10 min at 4 °C, carefully transfer 600 μl supernatant into a new 1.5 ml RNase-free tube.
4. Add equal volume (600 μl) 70% ethanol (see Recipes).
5. Add the mixed liquid into the HiBind^® RNA Mini Column (assembled 2 ml collection tube), centrifuge at 10,000 x g for 30 sec at 4 °C. Discard the filtrate and reuse the collection tube.
6. Add 300 µl RNA Wash Buffer I to the HiBind^® RNA Mini Column, centrifuge at 10,000 x g for 30 sec at 4 °C. Discard the filtrate and reuse the collection tube.
7. Add 500 µl RNA Wash Buffer I to the HiBind^® RNA Mini Column, centrifuge at 10,000 x g for 30 sec at 4 °C. Discard the filtrate and change a new 2 ml collection tube.
8. Add 500 μl RNA Wash Buffer II, centrifuge at 10,000 x g for 30 sec at 4 °C. Discard the filtrate and reuse the collection tube.
9. Repeat Step B8.
10. Centrifuge at 10,000 x g for 2 min to completely dry the HiBind^® RNA Mini Column.
11. Put the HiBind^® RNA Mini Column into a new 1.5 ml RNase-free tube, add 50 μl DNase/RNase-free ddH₂O into Column, and let it sit at room temperature for 1 min. Centrifuge at 10,000 x g for 1 min at 4 °C to elute RNA from the HiBind^® RNA Mini Column.
12. Measure RNA concentration using Nanodrop^TM 1000. Store the eluted RNA at -70 °C.
First-strand cDNA synthesis
Total RNA was reverse transcribed using PrimeScript^TM RT reagent Kit with gDNA Eraser (Perfect Real Time).
1. Remove gDNA reaction
  Reaction mixture:
  5x gDNA Eraser Buffer
  2 μl
  gDNA Eraser
  1 μl
  Total RNA
  700 ng (calculate volume)
  DNase/RNase-free ddH₂O
  up to 10 μl
  Reaction program:
  42 °C
  2 min
  4 °C
  ∞
2. Reverse transcription reaction
  Reaction mixture:
  The reaction solution of Step C1
  10 μl
  PrimeScript^TMRT Enzyme Mix I
  1 μl
  RT Primer Mix
  1 μl
  5x PrimeScript^TM Buffer 2 (for Real Time)
  4 μl
  DNase/RNase-free ddH₂O
  up to 20 μl
  Reaction program:
  37 °C
  15 min
  85 °C
  5 sec
  4 °C
  ∞
3. Dilute the obtained cDNA 20 times with 180 µl RNase-free H₂O.

Quantitative Real-time PCR (qRT-PCR)
qRT-PCR was performed using TB Green^TM Premix Ex Taq^TM Kit (Tli RNase H Plus):

qRT-PCR reaction mix:

2x TB Green^TM Premix Ex Taq^TM (Tli RNase H Plus)	10 μl
50x ROX Reference Dye II	0.4 μl
qRT-PCR Forward Primer	0.4 μl
qRT-PCR Reverse Primer	0.4 μl
The diluted first-strand cDNA	5 μl
DNase/RNase-free ddH₂O	up to 20 μl

Add the qRT-PCR reaction mix into each wall of MicroAmp^TM Optical 96-Well Reaction Plate with Barcode with three replicates for each sample. Seal the plate using MicroAmp^TM Optical Adhesive Film, and centrifuge the plate at room temperature for 3 min using the MPS 1000 Mini PCR Plate Spinner.
qRT-PCR program:
Stage 1:
Pre-denaturation at 95 °C for 30 sec
Stage 2:
PCR amplification (Reps: 40)

95 °C 5 sec

60 °C 34 sec
Stage 3:
Melting Curve

95 °C 15 sec

60 °C 1 min

95 °C 15 sec

Data analysis

Ct (cycle threshold) values were collected and exported from the Sequence Detection Software v1.4.0. Relative expression of marker genes in each sample was normalized against the internal control (Endo), such as the housekeeping genes NbActin or NbEF1α, and was calculated using the formula: 2^-ΔCT = 2^-(Ct_target ^{- Ct}_Endo⁾ calculated as ΔOD1-ΔOD2.
The relative expression level of marker genes in PAMP-treated samples to the control-treated sample was calculated using the following formula: 2^-ΔΔCT = 2^-(ΔCT_PAMP ^{- ΔCT}_control⁾ (Figure 3).

Figure 3. Transcription level changes of PTI marker gene CYP71D20 upon XEG1or flg22 treatment. Transcript levels were quantified by qRT-PCR and normalized to internal control NbEF-1α. Bars represent the mean fold changes (± S.E.M.) of the XEG1-treated or flg22-treated leaves relative to the value in control-treated leaves, which was set as 1.

Notes

It is important to use plants of similar size. Leaves of different plants should be collected at the same position with similar size.
The negative control should be included to measure the background level.
Cross-contamination of the PAMPs and negative control should be avoided during infiltration.
For each experiment, at least three replicates should be included for statistical analysis.
Different PAMPs may induce the defense-related marker gene expression at different time points.
The qRT-PCR program applies to Applied Biosystems 7500 fast Real-Time PCR System.

Recipes

1 mM flg22 stock solution
2.27 mg of flg22 (QRLSTGSRINSAKDDAAGLQIA) (Molecular Weight: 2272.52) was dissolved in 1 ml ddH₂O, and store at -20 °C. The stock was diluted to a final concentration of 200 nM
1 mM XEG1 stock solution
2.73 mg of XEG1 was dissolved in 1 ml ddH₂O, and store at -20 °C. The stock was diluted to a final concentration of 200 nM
70% ethanol
Add 30 ml DNase/RNase-free ddH₂O to 70 ml of absolute ethanol

Acknowledgments

This protocol was partially modified from the previously published paper by Wang et al. (2018). This work was supported by the China National Science Funds to Yuanchao Wang for Innovative Research Groups (Grant No. 31721004) and the funds to Yan Wang (Grant No. 31501622 and KJQN201663).

Competing interests

The authors declare that they have no conflicts of interest or competing interests.

References

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